PROJECT SUMMARY/ABSTRACT Helicobacter pylori colonizes the stomach of about half the human population worldwide and is the major pathogenic factor for peptic ulcer disease and chronic gastritis, as well as a major risk factor for gastric cancer and mucosa-associated lymphoid tissue lymphoma. H. pylori possess a cluster of polar flagella that the cell uses for motility, which is required for host colonization. A membranous sheath surrounds the H. pylori flagellum, a feature shared with other significant human pathogens, including pathogenic Vibrio species. Proposed functions for the flagellar sheath include protection of the flagellar filament from gastric acid and adherence to host cells. Nothing is known about the synthesis of the flagellar sheath in any bacterial species. Our preliminary studies identified an enzyme (ClsC) responsible for synthesis of the glycerophospholipid cardiolipin (CL) in H. pylori, and showed that clsC is required for flagellar biosynthesis in H. pylori G27. Highly purified H. pylori sheathed flagella contain substantial amounts of CL, confirming a functional link between ClsC and the flagellar sheath. These observations lead to our central hypothesis that CL is required for synthesis of the H. pylori flagellar sheath, and lesions in CL synthesis inhibit flagellar biosynthesis in H. pylori G27. A comparative genomics approach revealed a number of genes that are conserved in Helicobacter species that possess sheathed flagella but are absent in Helicobacter species that lack a flagellar sheath. Some of these genes encode an efflux system that our mutational analysis revealed to be required for flagellar biosynthesis in H. pylori G27. The efflux system may form a unique cage-like structure that surrounds the flagellar motor as disrupting one of the genes encoding the efflux system results in loss of the cage-like structure. These results led to our hypothesis that the efflux system transports CL to the outer membrane for assembly into the flagellar sheath. To understand the link between CL and biosynthesis of the H. pylori flagellar sheath, three Specific Aims will be pursued: (1) To define the role of CL in H. pylori flagellum biogenesis. (2) To determine if the efflux system is a CL transporter involved in flagellar sheath biosynthesis. (3) To determine if CL or the efflux system are required for localization of proteins to the flagellar sheath. The proposed research will address a critical gap in our knowledge of the biosynthesis of the H. pylori sheathed flagellum, as well as provide a paradigm for flagellar sheath biosynthesis in other medically relevant bacteria. The research will also have a broad impact on our knowledge of glycerophospholipid trafficking in the bacterial cell envelope.